The present invention relates generally to pyrrolidines, pharmaceutical compositions containing them, and their use as antagonists of urotensin II.
The integrated control of cardiovascular homeostasis is achieved through a combination of both direct neuronal control and systemic neurohormonal activation. Although the resultant release of both contractile and relaxant factors is normally under stringent regulation, an aberration in this status quo can result in cardiohemodynamic dysfunction with pathological consequences.
The principal mammalian vasoactive factors that comprise this neurohumoral axis, namely angiotensin-II, endothelin-1, norepinephrine, all function via an interaction with specific G-protein coupled receptors (GPCR). Urotensin-II, represents a novel member of this neurohumoral axis.
In the fish, this peptide has significant hemodynamic and endocrine actions in diverse end-organ systems and tissues:
smooth muscle contraction:
both vascular and non-vascular in origin including smooth muscle preparations from the gastrointestinal tract and genitourinary tract. Both pressor and depressor activity has been described upon systemic administration of exogenous peptide
osmoregulation:
effects which include the modulation of transepithelial ion (Na+, Clxe2x88x92) transport. Although a diuretic effect has been described, such an effect is postulated to be secondary to direct renovascular effects (elevated GFR)
metabolism:
urotensin-II influences prolactin secretion and exhibits a lipolytic effect in fish (activating triacylglycerol lipase resulting in the mobilization of non-esterified free fatty acids) (Pearson, et. al. Proc. Natl. Acad. Sci. (U.S.A.) 1980, 77, 5021; Conlon, et. al. J. Exp. Zool. 1996, 275, 226.)
In studies with human Urotensin-II it was found that it:
was an extremely potent and efficacious vasoconstrictor
exhibited sustained contractile activity that was extremely resistant to wash out
exhibited sustained contractile activity that was extremely resistant to wash out
had detrimental effects on cardiac performance (myocardial contractility)
Human Urotensin-II was assessed for contractile activity in the rat-isolated aorta and was shown to be the most potent contractile agonist identified to date. Based on the in vitro pharmacology and in vivo hemodynamic profile of human Urotensin-II it plays a pathological role in cardiovascular diseases characterized by excessive or abnormal vasoconstriction and myocardial dysfunction. (Ames et. al. Nature 1999, 401, 282) Compounds that antagonize the Urotensin-II receptor may be useful in the treatment of congestive heart failure, stroke, ischemic heart disease (angina, myocardial ischemia), cardiac arrhythmia, hypertension (essential and pulmonary), COPD, restenosis, asthma, (Hay D W P, Luttmann M A, Douglas S A: 2000, Br J Pharmacol: In press.) neurogenic inflammation and metabolic vasculopathies all of which are characterized by abnormal vasoconstriction and/or myocardial dysfunction. Since U-II and GPR14 are both expressed within the mammalian CNS (Ames et. al. Nature 1999, 401, 282), they also may be useful in the treatment of addiction, schizophrenia, impulsivity, anxiety, stress, depression, and neuromuscular function. Functional U-II receptors are expressed in rhabdomyosarcomas cell lines and therefore may have oncological indications. Urotensin may also be implicated in various metabolic diseases such as diabetes (Ames et. al. Nature 1999, 401, 282, Nothacker et al., Nature Cell Biology 1: 383-385, 1999)
In one aspect this invention provides for pyrrolidines and pharmaceutical compositions containing them.
In a second aspect, this invention provides for the use of pyrrolidines as antagonists of urotensin II, and as inhibitors of urotensin II.
In another aspect, this invention provides for the use of pyrrolidines for treating conditions associated with urotensin II imbalance.
In an yet another aspect, this invention provides for the use of these pyrrolidine analogs for the treatment of congestive heart failure, stroke, ischemic heart disease (angina, myocardial ischemia), cardiac arrhythmia, hypertension (essential and pulmonary), COPD, restenosis, asthma, neurogenic inflammation and metabolic vasculopathies, addiction, schizophrenia, impulsivity, anxiety, stress, depression, neuromuscular function, and diabetes.
The urotensin antagonist may be administered alone or in conjunction with one or more other therapeutic agents, said agents being selected from the group consisting of endothelin receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, vasopeptidase inhibitors, diuretics, digoxin, and dual non-selective xcex2-adrenoceptor and xcex11-adrenoceptor antagonists.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
The present invention provides for compounds of Formula I: 
wherein:
R1 is isobutyl, isopropyl, benzyl, carbamoylmethyl;
R2 is methyl, phenyl, naphthyl, indolyl, benzimidazolyl, quinolinyl, furanyl, thiopheneyl, pyridyl, benzofuranyl, or benzothiophenyl, substituted or unsubstituted by one or two halogen, methyl, methoxy, or methylenedioxy groups;
R3 is hydrogen, halogen, methyl, methoxy, nitro, or 2,3-(1,3-butadien-1,4-yl);
R4 is 3-dimethylaminopropyl, 2-(pyrrolidin-1-yl)ethyl, 2-(1-morpholino)ethyl, 2-(N-methyl anilino)ethyl, 2-(1-piperazinyl)ethyl, 2-(1-pyrrolidinonyl)ethyl, (1-methylpyrrolidin-2(S)-yl)methyl, 1-methylpyrrolidin-3(xc2x1)-yl, 1-benzylpiperldinyl-4-yl, (6-methylpyrid-2-yl)methyl, 3-dimethylamino benzyl, 2(S)-(aminocarbonyl)pyrrolidin-4(S)-yl, pyrrolidin-3(S)-yl, piperidin-4-yl, pyrrolidin-3(R)-yl, or cis-4-aminocyclohexyl; and
n is 1 or 2;
or a pharmacologically acceptable salt thereof.
When used herein, the terms xe2x80x98halogenxe2x80x99 and xe2x80x98haloxe2x80x99 include fluorine, chlorine, bromine and iodine and fluoro, chloro, bromo and iodo, respectively.
The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active form. All of these compounds and their diastereoisomers are contemplated to be within the scope of the present invention.
R1 is preferably isobutyl, isopropyl, or benzyl.
R2 preferably is phenyl, naphthyl, furanyl, thiopheneyl, benzofuranyl, or benzothiopheneyl, substituted or unsubstituted with one or two halogen, methyl, methoxy, or methylenedioxy groups.
R3 preferably is hydrogen, halogen, methyl, or methoxy.
R4 is preferably 3-dimethylaminopropyl, 2-(1-piperazinyl)ethyl, 2-(1-pyrrolidinonyl)ethyl, 1-methylpyrrolidin-3(xc2x1)-yl, 1-benzylpiperidin-4-yl, pyrrolidin-3(S)-yl, piperidin-4-yl, or pyrrolidin-3(R)-yl.
Preferred Compounds are:
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[4-iodo-3-(2-piperazin-1-yl-ethoxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[3-(3-dimethylamino-propoxy)-4-iodo-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[4-(3-dimethylamino-propoxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[4-(1-benzyl-piperidin-4-yloxy)-3-chloro-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[3-bromo-4-(3-dimethylamino-propoxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[3-bromo-4-(1-methyl-pyrrolidin-3-yloxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[4-(1-benzyl-piperidin-4-yloxy)-3-bromo-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzofuran-2-carboxylic acid ((S)-1-{(S)-1-[4-(3-dimethylamino-propoxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl)-amide;
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-[3-bromo-4-(piperidin-4yloxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl-amide; and
Benzo[b]thiophene-2-carboxylic acid ((S)-1-{(S)-1-[3-bromo-4-((R)-pyrrolidin-3-yloxy)-benzyl]-pyrrolidin-3-ylcarbamoyl}-3-methyl-butyl-amide.
Compounds of Formula (I) may be prepared as outlined in the following schemes: 
Resin-bound amine 3 was prepared by reductive amination of 2,6-dimethoxy-4-polystyrenebenzyloxy-benzaldehyde (DMHB resin) with N-nosylated diamine hydrochloride salt 2 which was prepared from 3(S)-(xe2x88x92)-(tert-butoxycarbonyl-amino)pyrrolidine (1) (Scheme 1). Reactions of resin-bound amine 3 with various amino acids, followed by removal of the protecting group, provided resin-bound amines 4. Amines 4 were then reacted with various acids to afford the corresponding resin-bound amides which were subsequently treated with potassium carbonate and thiophenol to give secondary amines 5. Reductive amination of resin-bound amines 5 with various hydroxy benzaldehydes produced resin-bound phenols 6. Phenols 6 were then reacted with various alcohols in the presence of triphenylphosphine and diisopropyl azodicarboxylate to give the corresponding resin-bound phenol ethers which were treated with 50% trifluoroacetic acid in 1,2-dichloroethane to afford targetted compounds 7. 
As shown in scheme 2, bis-nosylates 8 were synthesized from various commercially available or known hydroxyphenethyl alcohols. Phenols 9 were prepared by reactions of previously synthesized amines 5 (scheme 1) with bis-nosylates 8 in the presence of tetrabutylammonium iodide, followed by hydrolysis of the resulting mononosylate intermediates. Phenols 9 were then reacted with various alcohols in the presence of triphenylphosphine and diisopropyl azodicarboxylate to give the corresponding resin-bound phenol ethers which were treated with 50% trifluoroacetic acid in 1,2-dichloroethane to afford targetted compounds 10.
In order to use a compound of the Formula (I) or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Compounds of Formula (I) and their pharmaceutically acceptable salts may be administered in a standard manner for the treatment of the indicated diseases, for example orally, parenterally, sub-lingually, transdermally, rectally, via inhalation or via buccal administration.
Compounds of Formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, agar, pectin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of the compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (1) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogues.
Typical transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer to themselves a single dose.
Each dosage unit for oral administration contains suitably from 0.1 mg to 500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. Each dosage unit for intranasal administration contains suitably 1-400 mg and preferably 10 to 200 mg per person. A topical formulation contains suitably 0.01 to 1.0% of a compound of Formula (I).
The daily dosage regimen for oral administration is suitably about 0.01 mg/Kg to 40 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. The daily dosage regimen for parenteral administration is suitably about 0.001 mg/Kg to 40 mg/Kg, of a compound of the Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. The daily dosage regimen for intranasal administration and oral inhalation is suitably about 10 to about 500 mg/person. The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity.
These pyrrolidine analogs may be used for the treatment of congestive heart failure, stroke, ischemic heart disease (angina, myocardial ischemia), cardiac arrhythmia, hypertension (essential and pulmonary), COPD, restenosis, asthma, neurogenic inflammation and metabolic vasculopathies, addiction, schizophrenia, impulsivity, anxiety, stress, depression, neuromuscular function, and diabetes.
The urotensin antagonist may be administered alone or in conjunction with one or more other therapeutic agents, said agents being selected from the group consisting of endothelin receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, vasopeptidase inhibitors, diuretics, digoxin, and dual non-selective xcex2-adrenoceptor and xcex11-adrenoceptor antagonists.
No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated by the following tests:
Radioligand Binding
HEK-293 cell membranes containing stable cloned human and rat GPR-14 (20 ug/assay) were incubated with 200 pM [125I] h-U-II (200 Ci/mmolxe2x88x921 in the presence of increasing concentrations of test compounds in DMSO (0.1 nM to 10 uM), in a final incubation volume of 200 ul (20 mM Tris-HCl, 5 mM MgCl2). Incubation was done for 30 minutes at room temperature followed by filtration GF/B filters with Brandel cell harvester. 125I labeled U-II binding was quantitated by gamma counting. Nonspecific binding was defined by 125I U-II binding in the presence of 100 nM of unlabeled human U-II. Analysis of the data was performed by nonlinear least square fitting.
Ca2+-mobilization
A microtitre plate based Ca2+-mobilization FLIPR assay (Molecular Devices, Sunnyvale, Calif.) was used for the functional identification of the ligand activating HEK-293 cells expressing (stable) recombinant GPR-14. The day following transfection, cells were plated in a poly-D-lysine coated 96 well black/clear plates. After 18-24 hours the media was aspirated and Fluo 3AM-loaded cells were exposed to various concentrations (10 nM to 30 uM) of test compounds followed by h-U-II. After initiation of the assay, fluorescence was read every second for one minute and then every 3 seconds for the following one minute. The inhibitory concentration at 50% (IC50)was calculated for various test compounds.
Inositol Phosphates Assays
HEK-293-GPR14 cells in T150 flask were prelabeled overnight with 1 uCi myo-[3H] inositol per ml of inositol free Dulbecco""s modified Eagel""s medium. After labeling, the cells were washed twice with Dulbecco""s phosphate-buffered saline (DPBS) and then incubated in DPBS containing 10 mM LiCl for 10 min at 37xc2x0 C. The experiment was initiated by the addition of increasing concentrations of h-U-II (1 pM to 1 xcexcM ) in the absence and presence of three different concentrations (0.3, 1 and 10 uM) of test compounds and the incubation continued for an additional 5 min at 37xc2x0 C. after which the reaction was terminated by the addition of 10% (final concentration) trichloroacetic acid and centrifugation. The supernatants were neutralized with 100 ul of 1M Trizma base and the inositol phosphates were separated on AG 1-X8 columns (0.8 ml packed, 100-200 mesh) in formate phase. Inositol monophosphate was eluted with 8 ml of 200 mM ammonium formate. Combined inositol di and tris phosphate was eluted with 4 ml of 1M ammonium formate/0.1 M formic acid. Eluted fractions were counted in beta scintillation counter. Based on shift from the control curve KB was calculated.
Activity for the compounds of this invention range from (radioligand binding assay): Ki=1 nM-10000 nM (example 15 Ki=390 nM)